Nearly two-thirds of all hospital-acquired infections (HAIs) are caused by Staphylococcus epidermidis and S. aureus. These Staphylococci are particularly troublesome due to their propensity to form biofilms, which are communities of bacteria that adhere to a substrate and form microcolonies, typically surrounded by a polysaccharide matrix. Recurrent bacterial infections caused by biofilms are refractory to antibiotic treatment and host immune responses and thus must often be surgically removed. Recent work has implicated a single protein family in the formation of staphylococcal biofilms. These proteins, called Aap in S. epidermidis and SasG in S. aureus, contain up to 17 tandem G5 domain repeats in the B-repeat region that are necessary and sufficient for the formation of staphylococcal biofilms. We have shown that G5 domains act as zinc-dependent adhesion modules that trigger formation of a "zinc zipper" adhesive structure between staphylococcal cells in biofilms. Chelation of zinc from the media inhibits biofilm formation by both S. epidermidis and S. aureus, including several MRSA strains. We have solved the crystal structure of a short consensus B-repeat construct, revealing a highly elongated "pleated ribbon" fold that forms an anti-parallel dimer in the presence of zinc. We are now studying longer B-repeat constructs that match the length required for biofilm formation in vivo. In the presence of zinc or copper, these constructs form fibrous aggregates with amyloid-like characteristics. We will define the molecular mechanism for Aap-mediated intercellular adhesion and amyloid formation in staphylococcal biofilms. In addition to crystal structures of the short consensus repeat complexed with zinc and copper, we will determine the structure of longer B-repeat constructs by a combination of techniques. Finally, we will analyze the role of Aap in the formation of cultured staphylococcal biofilms, and compare its function with that of the icaADBC operon responsible for biosynthesis of the biofilm polysaccharide. The feasibility of these studies is enhanced by our strong published and preliminary data, the reagents we have developed, our success in solving the crystal structure of the short consensus repeat, and our expertise in biophysical and crystallographic approaches. We will also benefit from a close collaboration with Dr. Dan Hassett, an expert in bacterial genetics, biochemistry, and biofilms, as well as interactions with key leaders in cryo-electron microscopy, amyloid formation, and analytical ultracentrifugation. The interdisciplinary nature of this grant will allow us to test our hypotheses by experimental biophysical approaches as well as clinically-relevant biofilm experiments. Relevance: Hospital-acquired infections (HAIs) are the fourth-leading cause of death in the United States, leading to over 100,000 deaths per year. Staphylococci cause nearly two-thirds of these HAIs. The propensity of Staphylococci to form biofilms leads to recurrent, recalcitrant infections. These studies will lead to a better understanding of a major mechanism for biofilm formation and will help aid the development of inexpensive and broadly effective approaches for preventing biofilm formation. PUBLIC HEALTH RELEVANCE: Biofilms are communities of bacteria that attach to a surface and become highly resistant to antibiotics or immune responses;biofilms can cause recurrent, hard-to-treat infections, particularly with implanted medical devices. In this proposal we will investigate the details of how Staphylococcus bacterial cells stick to one another in a biofilm, and how they become resistant to physical or chemical challenges. The results from this research will provide new approaches for preventing biofilm formation or potentially reversing pre-formed biofilms.